New genetic models and mesenchymal cells as tools to ameliorate anti-tumor immunity

Résumé Le but final de ce projet est d'utiliser des cellules T ou des cellules souches mésenchymateuses modifiées génétiquement afin de surexprimer localement les deux chémokines CXCL13 et CCL2 ensemble ou chacune séparément à l'intérieur d'une tumeur solide. CXCL13 est supposé induire des structures lymphoïdes ectopiques. Un niveau élevé de CCL2 est présumé initier une inflammation aiguë. La combinaison des deux effets amène à un nouveau modèle d'étude des mécanismes régulateur de la tolérance périphérique et de l'immunité tumorale. Les connaissances acquises grâce à ce modèle pourraient permettre le développement ou l'amélioration des thérapies immunes du cancer. Le but premier de ce travail a été l'établissement d'un modèle génétique de la souris permettant d'exprimer spécifiquement dans la tumeur les deux chémokines d'intérêt à des niveaux élevés. Pour accomplir cette tâche, qui est en fait une thérapie génétique de tumeurs solides, deux types de cellules porteuses potentielles ont été évaluées. Des cellules CD8+ T et des cellules mésenchymateuses de la moelle osseuse transférées dans des receveurs portant une tumeur. Si on pouvait répondre aux besoins de la thérapie génétique, indépendamment de la thérapie immune envisagée, on posséderait là un outil précieux pour bien d'autres approches thérapeutiques. Plusieurs lignées de souris transgéniques ont été générées comme source de cellules CD8+ T modifiées afin d'exprimer les chémokines d'intérêt. Dans une approche doublement transgénique les propriétés de deux promoteurs spécifiques de cellules T ont été combinées en utilisant la technologie Cre-loxP. Le promoteur de granzyme B confère une dépendance d'activation et le promoteur distal de lck assure une forte expression constitutive dès que les cellules CD8+ T ont été activées. Les transgènes construits ont montré une bonne performance in vivo et des souris qui expriment CCL2 dans des cellules CD8+ T activées ont été obtenues. Ces cellules peuvent maintenant être utilisées avec différents protocoles pour transférer des cellules T cytotoxiques (CTL) dans des receveurs porteur d'une tumeur, permettant ainsi d'évaluer leur capacité en tant que porteuse de chémokine d'infiltrer la tumeur. L'établissement de souris transgéniques, qui expriment pareillement CXCL13 est prévu dans un avenir proche. L'évaluation de cellules mésenchymateuses de la moelle osseuse a démontré que ces cellules se greffent efficacement dans le stroma tumoral suite à la co-injection avec des cellules tumorales. Cela représente un outil précieux pour la recherche, vu qu'il permet d'introduire des cellules manipulées dans un modèle tumoral. Les résultats confirment partiellement d'autres résultats rapportés dans un modèle amélioré. Cependant, l'efficacité et la spécificité suggérées de la migration systémique de cellules mésenchymateuses de la moelle osseuse dans une tumeur n'ont pas été observées dans notre modèle, ce qui indique, que ces cellules ne se prêtent pas à une utilisation thérapeutique. Un autre résultat majeur de ce travail est l'établissement de cultures de cellules mésenchymateuses de la moelle osseuse in vitro conditionnées par des tumeurs, ce qui a permis à ces cellules de s'étendre plus rapidement en gardant leur capacité de migration et de greffe. Cela offre un autre outil précieux, vu que la culture in vitro est un pas nécessaire pour une manipulation thérapeutique. Abstract The ultimate aim of the presented project is to use genetically modified T cells or mesenchymal stem cells to locally overexpress the two chemokines CXCL13 and CCL2 together or each one alone inside a solid tumor. CXCL13 is supposed to induce ectopic lymphoid structures and a high level of CCL2 is intended to trigger acute inflamation. The combination of these two effects represents a new model for studying mechanisms that regulate peripheral tolerance and tumor immunity. Gained insights may help developing or improving immunotherapy of cancer. The primary goal of the executed work was the establishment of a genetic mouse model that allows tumor-specific expression of high levels of the two chemokines of interest. For accomplishing this task, which represents gene therapy of solid tumors, two types of potentially useful carrier cells were evaluated. CD8+ T cells and mesenchymal bone marrow cells to be used in adoptive cell transfers into tumor-bearing mice. Irrespectively of the envisaged immunotherapy, satisfaction of so far unmet needs of gene therapy would be a highly valuable tool that may be employed by many other therapeutic approaches, too. Several transgenic mouse lines were generated as a source of CD8+ T cells modified to express the chemokines of interest. In a double transgenic approach the properties of two T cell-specific promoters were combined using Cre-loxP technology. The granzyme B promoter confers activation-dependency and the lck distal promoter assures strong constitutive expression once the CD8+ T cell has been activated. The constructed transgenes showed a good performance in vivo and mice expressing CCL2 in activated CD8+ T cells were obtained. These cells can now be used with different protocols for adoptively transferring cytotoxic T cells (CTL) into tumor-bearing recipients, thus allowing to study their capacity as tumor-infiltrating chemokine carrier. The establishment of transgenic mice likewisely expressing CXCL13 is expected in the near future. In addition, T cells from generated single transgenic mice that have high expression of an EGFP reporter in both CD4+ and CD8+ cells can be easily traced in vivo when setting up adoptive transfer conditions. The evaluation of mesenchymal bone marrow cells demonstrated that these cells can efficiently engraft into tumor stroma upon local coinjection with tumor cells. This represents a valuable tool for research purposes as it allows to introduce manipulated stromal cells into a tumor model. Therefore, the established engraftment model is suited for studying the envisaged immunotherapy. These results confirm to some extend previously reported results in an improved model, however, the suggested systemic tumor homing efficiency and specificity of mesenchymal bone marrow cells was not observed in our model indicating that these cells may not be suited for therapeutic use. Another major result of the presented work is the establishment oftumor-conditioned in vitro culture of mesenchymal bone marrow cells, which allowed to more rapidly expand these cells while maintaining their tumor homing and engrafting capacities. This offers another valuable tool as in vitro culture is a necessary step for therapeutic manipulations.

Development and Preliminary Evaluation of a Rapid Oligochromatographic Assay for Specific Detection of New Human Influenza A H1N1 Virus

A new oligochromatographic assay, Speed-Oligo Novel Influenza A H1N1, was designed and optimized for the specific detection of the 2009 influenza A H1N1 virus. The assay is based on a PCR method coupled to detection of PCR products by means of a dipstick device. The target sequence is a 103-bp fragment within the hemagglutinin gene. The analytical sensitivity of the new assay was measured with serial dilutions of a plasmid that contained the target sequence, and we determined that down to one copy per reaction of the plasmid was reliably detected. Diagnostic performance was assessed with 103 RNAs from suspected cases (40 positive and 63 negative results) previously analyzed with a reference real-time PCR technique. All positive cases were confirmed, and no false-positive results were detected with the new assay. No cross-reactions were observed when other viral strains or clinical samples with other respiratory viruses were tested. According to these results, this new assay has 100% sensitivity and specificity. The turnaround time for the whole procedure was 140 min. The assay may be especially useful for the specific detection of 2009 H1N1 virus in laboratories not equipped with real-time PCR instruments

Organisation des secours extrahospitaliers lors du sommet 2003 du G8: nouveau concept de poste médical mobile (PMM) [Prehospital medical care organization during the 2003 G8 summit: a new concept of Mobile Medical Squadrons (MMS)].

OBJECTIVE: The occurrence of the 2003 G8 summit in Evian and the threat of major civil riots or even terrorist attacks in the Swiss neighbourhood forced us to imagine a new system of rescue and medical care in case of numerous victims. Previous occurrences of the G8 in Europe or America have demonstrated the need of flexible and mobile structures, able to respond quickly to crowd movements, unlike the usual static structure of rescue systems designed for major accidents. METHODS: We developed a new concept of Mobile Medical Squadrons (MMS) consisting of several vehicles and medical care and rescue human resources. In our concept, each MMS consisted of 3 emergency doctors, 5 paramedics and 9 first-aid workers. They were designed to handle 15 patients, with a large autonomy in terms of rescue, medical care, evacuation and medical authority. The equipment included medical, resuscitation, simple decontamination, evacuation and communication materials. RESULTS: The MMS were dispatched four times during the G8 summit following civil riots. They took care of 12 injured patients. CONCLUSION: The concept of MMS as a reinforcement of the existing rescue and health care resources appears as a new flexible, a modular and useful concept for the medical management of collective prehospital emergency situations. Its use is suggested instead of the traditional static concept of rescue systems designed for major accidents.

Evidence of new risk genetic factor to systemic lupus erythematosus: the UBASH3A gene

The ubiquitin associated and Src-homology 3 (SH3) domain containing A (UBASH3a) is a suppressor of T-cell receptor signaling, underscoring antigen presentation to T-cells as a critical shared mechanism of diseases pathogenesis. The aim of the present study was to determine whether the UBASH3a gene influence the susceptibility to systemic lupus erythematosus (SLE) in Caucasian populations. We evaluated five UBASH3a polymorphisms (rs2277798, rs2277800, rs9976767, rs13048049 and rs17114930), using TaqMan® allelic discrimination assays, in a discovery cohort that included 906 SLE patients and 1165 healthy controls from Spain. The SNPs that exhibit statistical significance difference were evaluated in a German replication cohort of 360 SLE patients and 379 healthy controls. The case-control analysis in the Spanish population showed a significant association between the rs9976767 and SLE (Pc = 9.9E-03 OR = 1.21 95%CI = 1.07-1.37) and a trend of association for the rs2277798 analysis (P = 0.09 OR = 0.9 95%CI = 0.79-1.02). The replication in a German cohort and the meta-analysis confirmed that the rs9976767 (Pc = 0.02; Pc = 2.4E-04, for German cohort and meta-analysis, respectively) and rs2277798 (Pc = 0.013; Pc = 4.7E-03, for German cohort and meta-analysis, respectively) UBASH3a variants are susceptibility factors for SLE. Finally, a conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs9976767 polymorphism. Our results suggest that UBASH3a gene plays a role in the susceptibility to SLE. Moreover, our study indicates that UBASH3a can be considered as a common genetic factor in autoimmune diseases.

Rhodnius barretti, a new species of Triatominae (Hemiptera: Reduviidae) from western Amazonia

Rhodnius barretti , a new triatomine species, is described based on adult specimens collected in rainforest environments within the Napo ecoregion of western Amazonia (Colombia and Ecuador). R. barretti resembles Rhodnius robustus s.l. , but mitochondrial cytochrome b gene sequences reveal that it is a strongly divergent member of the “robustus lineage”, i.e., basal to the clade encompassing Rhodnius nasutus , Rhodnius neglectus , Rhodnius prolixus and five members of the R. robustus species complex. Morphometric analyses also reveal consistent divergence from R. robustus s.l. , including head and, as previously shown, wing shape and the length ratios of some anatomical structures. R. barretti occurs, often at high densities, in Attalea butyracea and Oenocarpus bataua palms. It is strikingly aggressive and adults may invade houses flying from peridomestic palms. R. barretti must therefore be regarded as a potential Trypanosoma cruzi vector in the Napo ecoregion, where Chagas disease is endemic.

Living with digestive stomas: strategies to cope with the new bodily reality.

OBJECTIVE the objective in this study was to describe the strategies developed by digestive stoma patients to cope with their situation. METHOD a qualitative and descriptive study was undertaken, involving 21 stoma patients living in the provinces of Málaga and Granada (Spain). The informants were selected in accordance with criteria of appropriateness and diversity, through intentional sampling. The data were collected by means of semistructured interviews. RESULTS the content analysis revealed three categories around which the distinct strategies were developed: Self-care, Adaptation to the bodily change and Self-help. CONCLUSION the strategies developed are focusing on achieving the effective management of the stoma and are closely linked with the achievement of autonomy. Discovering the strategies applied is fundamental for the nursing professionals to offer high-quality care, centered on people and their process.

A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease

A pseudogene, designated as "ps(5.8S+ITS-2)", paralogous to the 5.8S gene and internal transcribed spacer (ITS)-2 of the nuclear ribosomal DNA (rDNA), has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2)" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2)" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.

QFT control for vibration reduction in structures equipped with MR dampers

This short paper addresses the problem of designing a QFT (quantitative feedback theory) based controllers for the vibration reduction in a 6-story building structure equipped with shear-mode magnetorheological dampers. A new methodology is proposed for characterizing the nonlinear hysteretic behavior of the MR damper through the uncertainty template in the Nichols chart. The design procedure for QFT control design is briefly presented

X-ray structures of Sap1 and Sap5: structural comparison of the secreted aspartic proteinases from Candida albicans.

Proteolytic activity is an important virulence factor for Candida albicans (C. albicans). It is attributed to the family of the secreted aspartic proteinases (Saps) from C. albicans with a minimum of 10 members. Saps show controlled expression and regulation for the individual stages of the infection process. Distinct isoenzymes can be responsible for adherence and tissue damage of local infections, while others cause systemic diseases. Earlier, only the structures of Sap2 and Sap3 were known. In our research, we have now succeeded in solving the X-ray crystal structures of the apoenzyme of Sap1 and Sap5 in complex with pepstatin A at 2.05 and 2.5 A resolution, respectively. With the structure of Sap1, we have completed the set of structures of isoenzyme subgroup Sap1-3. Of subgroup Sap4-6, the structure of the enzyme Sap5 is the first structure that has been described up to now. This facilitates comparison of structural details as well as inhibitor binding modes among the different subgroup members. Structural analysis reveals a highly conserved overall secondary structure of Sap1-3 and Sap5. However, Sap5 clearly differs from Sap1-3 by its electrostatic overall charge as well as through structural conformation of its entrance to the active site cleft. Design of inhibitors specific for Sap5 should concentrate on the S4 and S3 pockets, which significantly differ from Sap1-3 in size and electrostatic charge. Both Sap1 and Sap5 seem to play a major part in superficial Candida infections. Determination of the isoenzymes' structures can contribute to the development of new Sap-specific inhibitors for the treatment of superficial infections with a structure-based drug design program.

New insights into the role of the immune microenvironment in breast carcinoma.

Recently, immune edition has been recognized as a new hallmark of cancer. In this respect, some clinical trials in breast cancer have reported imppressive outcomes related to laboratory immune findings, especially in the neoadjuvant and metastatic setting. Infiltration by tumor infiltrating lymphocytes (TIL) and their subtypes, tumor-associated macrophages (TAM) and myeloid-derived suppressive cells (MDSC) seem bona fide prognostic and even predictive biomarkers, that will eventually be incorporated into diagnostic and therapeutic algorithms of breast cancer. In addition, the complex interaction of costimulatory and coinhibitory molecules on the immune synapse and the different signals that they may exert represent another exciting field to explore. In this review we try to summarize and elucidate these new concepts and knowledge from a translational perspective focusing on breast cancer, paying special attention to those aspects that might have more significance in clinical practice and could be useful to design successful therapeutic strategies in the future.

New insights into pathophysiology of vestibular migraine.

Vestibular migraine (VM) is a common disorder in which genetic, epigenetic, and environmental factors probably contribute to its development. The pathophysiology of VM is unknown; nevertheless in the last few years, several studies are contributing to understand the neurophysiological pathways involved in VM. The current hypotheses are mostly based on the knowledge of migraine itself. The evidence of trigeminal innervation of the labyrinth vessels and the localization of vasoactive neuropeptides in the perivascular afferent terminals of these trigeminal fibers support the involvement of the trigemino-vascular system. The neurogenic inflammation triggered by activation of the trigeminal-vestibulocochlear reflex, with the subsequent inner ear plasma protein extravasation and the release of inflammatory mediators, can contribute to a sustained activation and sensitization of the trigeminal primary afferent neurons explaining VM symptoms. The reciprocal connections between brainstem vestibular nuclei and the structures that modulate trigeminal nociceptive inputs (rostral ventromedial medulla, ventrolateral periaqueductal gray, locus coeruleus, and nucleus raphe magnus) are critical to understand the pathophysiology of VM. Although cortical spreading depression can affect cortical areas involved in processing vestibular information, functional neuroimaging techniques suggest a dysmodulation in the multimodal sensory integration and processing of vestibular and nociceptive information, resulting from a vestibulo-thalamo-cortical dysfunction, as the pathogenic mechanism underlying VM. The elevated prevalence of VM suggests that multiple functional variants may confer a genetic susceptibility leading to a dysregulation of excitatory-inhibitory balance in brain structures involved in the processing of sensory information, vestibular inputs, and pain. The interactions among several functional and structural neural networks could explain the pathogenic mechanisms of VM.

Robust control of structures subject to uncertain disturbances and actuator dynamics

The objective the present research is try to find some control design strategies, which must be effective and closed to the real operation conditions. As a novel contribution to structural control strategies, the theories of Interval Modal Arithmetic, Backstepping Control and QFT (Qualitative Feedback Theory) will be studied. The steps to follow are to develop first new controllers based on the above theories and then to implement the proposed control strategies to different kind of structures. The report is organized as follows. The Chapter 2 presents the state-of-the-art on structural control systems. The chapter 3 presents the most important open problems found in field of structural control. The exploratory work made by the author, research proposal and working plan are given in the Chapter 4

Conflict resolution and evolution of social structures in insect societies

In colonies of social Hymenoptera (which include all ants, as well as some wasp and bee species), only queens reproduce whereas workers generally perform other tasks. The evolution of worker's reproductive altruism can be explained by kin selection, which states that workers can indirectly transmit copies of their genes by helping the reproduction of relatives. The relatedness between queens and workers may however be low, particularly when there are multiple queens per colony, which limits the transmission of copies of workers genes and increases potential conflicts between colony members. In this thesis, we investigated the link between social structure variations and conflicts, and explored the mechanisms involved in variation of colony queen number in ants. According to kin selection, workers should rear the brood they are most related to. In social Hymenoptera, males are haploid whereas females (workers and queens) are diploid. As a result, workers can be up to three times more related to females than males in some colonies, where they should consequently favour the production of females. In contrast, queens are equally related to daughters and sons in all types of colonies and therefore should favour a balanced sex ratio. In a meta-analysis across all studies of social Hymenoptera, we showed that colony sex ratio is generally largely influenced by workers. Hence, the evolution of social structures where queens and workers are equally related to males and females may contribute to decrease the conflict between the two castes over colony sex ratio. Another conflict between queens and workers can occur over male production. Many species contain workers that still have the ability to lay haploid eggs. In some social structures, workers are on average more related to sons of queens than to sons of other workers. As a result, workers should eliminate worker-laid eggs to favour queen-laid eggs. We showed that in the ant Formica selysi, workers eliminate more worker-laid than queen-laid eggs, independently of colony social structure. These results therefore suggest that worker policing can evolve independently from relatedness, potentially because of costs of worker reproduction at the colony-level. Colony queen number is a key parameter that influences relatedness between group members. Queen body size is generally linked to the success of independent colony foundation by single queens and may influence the number of queens in the new colony. In the ant F. selysi, single-queen colonies produce larger queens than multiple-queen colonies. We showed that this association results from genes or maternal effects transmitted to the eggs. However, we also found that queens produced in colonies of the two social forms did not differ in their general ability to found new colonies independently. Queen body size may also influence queen dispersal ability and constrain small queens to be re-adopted in their original nest after mating at proximity. We tested the acceptance of new queens in another ant species, Formica paralugubris, which has numerous queens per colony. Our results show that workers do not discriminate between nestmate and foreign queens, and more generally accept new queens at a limited rate. To conclude, this thesis shows that mechanisms influencing variation in colony queen number and the influence of these changes on conflict resolution are complex. Data gathered in this thesis therefore constitute a solid background for further research on the evolution and the maintenance of complex organisations in insect societies.

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

BACKGROUND: The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. RESULTS: We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. CONCLUSIONS: "RodCell" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. AVAILABILITY: RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html, (after acceptance of the publication).

In vivo visualization of F-actin structures during the development of the moss Physcomitrella patens.

* The 'in planta' visualization of F-actin in all cells and in all developmental stages of a plant is a challenging problem. By using the soybean heat inducible Gmhsp17.3B promoter instead of a constitutive promoter, we have been able to label all cells in various developmental stages of the moss Physcomitrella patens, through a precise temperature tuning of the expression of green fluorescent protein (GFP)-talin. * A short moderate heat treatment was sufficient to induce proper labeling of the actin cytoskeleton and to allow the visualization of time-dependent organization of F-actin structures without impairment of cell viability. * In growing moss cells, dense converging arrays of F-actin structures were present at the growing tips of protonema cell, and at the localization of branching. Protonema and leaf cells contained a network of thick actin cables; during de-differentiation of leaf cells into new protonema filaments, the thick bundled actin network disappeared, and a new highly polarized F-actin network formed. * The controlled expression of GFP-talin through an inducible promoter improves significantly the 'in planta' imaging of actin.